Many advances in proteomics have been driven by the development of mass spectrometric-based technologies and tools1. Although mass spectrometry (MS) was invented in the early 1900s for the detection of small molecules, a quantum leap was achieved in the late 1980s when Fenn and Tanaka showed independently that large biomolecules (proteins, deoxy ribonucleic acid (DNA), etc) can be detected and quantitated accurately by MS. Fenn's technique called Electro Spray Ionization (ESI) nebulizes a protonated liquid into a fine spray using a high voltage prior to MS detection2. Tanaka's method called Matrix Assisted Laser Desorption Ionization (MALDI) utilizes a high energy absorbing molecule to desorb intact proteins on a solid inert surface3. A flavor of this latter technique, called Surface Enhanced Laser Desorption Ionization (SELDI) permits the immobilization of molecules on different active surfaces. SELDI is described in U.S. Pat. No. 5,719,0604, U.S. Pat. No. 6,225,0475, and in Weinberger et al., 20006.
A number of reports have appeared over the past few years regarding proteomic profiling with SELDI-TOF technology, in combination with artificial intelligence7. Reported sensitivities and specificities with the technique for ovarian, prostate, and breast cancer diagnoses are better than those obtained with current serologic cancer biomarkers8. Also, the technique is reported to detect early as well as late stage disease with similar efficiency, thus offering a potentially powerful new cancer screening tool9.
Extremely better specificies and sensitivities are obtained if the SELDI chipholder is analysed by a high resolution mass spectrometer (e.g. ABI/Sciex QSTAR ms)10. A Tandem MS interface, model PCI-1000 is available from Ciphergen Biosystens for such purposes.
The combination of techniques such as polyacrylamide gel electrophoresis (PAGE)7,8, reverse phase high performance liquid chromatography (RP-HPLC)9-11, affinity capture12,13 and protein chips14 with mass spectrometry (MS) has provided a series of important tools for the investigation of numerous facets of proteomics. The identification and characterization of the chemical features of proteins are essential prerequisites for understanding the dynamics and connectivity of their interactions as well as the diversity of their biological functions in living organisms. As a common method, peptide mass fingerprinting (PMF) identifies proteins by comparing the peptide mass fingerprint obtained from mass spectrometry analysis of enzymatic (or chemical) digestions to mass profiles generated by in-silico digestion of proteins15. This approach requires relatively purified target protein and is often used with protein fractionation techniques. Prior to enzymatic or chemical digestion, proteins are denatured, reduced and alkylated. Digestion is generally performed overnight to ensure complete cleavage. Structural characterization of proteins becomes all the more difficult if one considers that the vast majority of proteins contain disulfide bridges, phosphorylation, glycosylation sites or a combination of the above. Another less popular but more powerful method than PMF is the analysis of ms/ms product ion spectra to determine the peptide backbone fragmentation.
Thus, to study biological systems at the protein level, efforts have been directed at improvements in instrumentation and the development of novel technologies.
Protein chip array technology is based on two powerful techniques: chromatography and mass spectroscopy. It consists of selective protein extraction, retention and enrichment of proteins on chromatographic chip surfaces and their subsequent analysis by mass spectroscopy. The protein chip array surfaces function as a solid phase extraction media that support isolation and clean up of analytes prior to mass spectroscopic investigation.
By comparing samples between control and experimental groups or between healthy and diseased individuals, in one use of the technology, protein chip array profiling allows the rapid creation of phenotypic fingerprints and the identification of biomarkers of particular metabolic or disease states.
Thus, together with the growth of this technology comes the need for protein chemistry techniques that are applicable to protein chips. Three groups have reported a single on-chip reaction prior to MS analysis. Pentafluoropropionic acid and trifluoroacetic acid (TFA) were used to perform limited acid hydrolysis of proteins using a vapor-phase hydrolysis procedure16. The method was proposed to generate peptide ladders indicating primary sequences. However, side reactions, such as oxidation of methionine residues and deamidation of asparagine or glutamine, were systematically observed16. A second group reported a procedure for the identification of parvalbumin alpha (PVA) using on-chip enzymatic proteolysis17. Four peptides were identified after a 2-hour digestion and nine peptides were identified after 18 hours. PVA is an 11.85 kilodalton (kDa) linear N-terminus acetylated polypeptide, which is not representative of most of the proteins in existing proteomes as it lacks complex modifications such as disulfide bridges, phosphorylated or glycosylated moieties. Finally, an on-chip tryptic digestion method has been applied to recombinant prolactin-inducible protein (PIP). This purified 16.57 kDa protein has two disulfide bridges and one N-glycosylation site18.
In all the above examples, most chemical and enzymatic steps were carried out in solution. Relatively simple proteins were tested, and in all cases, a single on-chip step of treatment was performed. On-chip protein denaturation, reduction, alkylation, deglycosylation and dephosphorylation using protein chips have not been previously reported. In addition, previous reports have generally been based on rather simple proteins.
Thus, there remains a need for improved methods allowing structural characterization of proteins.
There further remains a need for methods of protein identification, which reduce sample loss, enable rapid and sensitive detection and identification of proteins with minimal sample manipulation.
There also remains a need for simple methods allowing complete on-chip chemistry (including enzymatic treatment) and characterization of proteins.
The present invention seeks to meet these needs and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.